1. Field of the Invention
The present invention relates generally to the fields of molecular biology, biochemistry and medical therapy. More specifically, the present invention relates to major renal receptors for low molecular weight proteins and potential uses of the receptors for therapy to prevent renal toxicity.
2. Description of the Related Art
Countless proteins of small or intermediate molecular weight, filtered freely or partially through the renal glomerulus, are bound by scavenger pathway receptors on the luminal surface of proximal tubular cells for reuptake (Batuman et al., 1990; Birn et al., 1997; Christensen et al., 1995; Saito et al., 1994). These proteins are then transcytosed back into the circulation, or degraded, releasing amino acids for fresh protein synthesis. The scavenger pathway receptors of the proximal tubular are an essential physiological defense against the urinary loss of a diverse array of plasma proteins essential to homeostatic functions from coagulation to lipid metabolism. Unfortunately, exposure of the scavenger receptors to unusually high concentrations of ligands due to overproduction of a ligand such as myeloma light chains introduction of a freely filtered drug such a s gentamicin, or increased glomerular permeability can disrupt the physiological balance, resulting in severe nephrotoxicity.
The relative contribution of proximal and distal elements to the development of protein nephrotoxicity remains controversial and ill-defined. For light chain nephrotoxicity if they fail to be reabsorbed proximally, the ligands are delivered into the distal tubular segments of the nephron, where they precipitate as casts in combination with Tamm-Horsfall protein (Huang et al., 1997; Weiss et al., 1981; Winearls, 1995), the severity of the renal dysfunctional correlates with the degree of (distal) cast formation (Myatt, 1994; Winearls, 1995). However, some light chains are associated with a pure (proximal) Fanconi syndrome. Myoglobin on the other hand is associated with little (distal) cast formation, but marked proximal tubular damage, with clinical acute tubular necrosis (Paller, 1988; Zager, 1991).
Immunoglobulin light chains are filtered at the glomerulus and endocytosed in the proximal tubule (Batuman et al., 1990; Batuman et al., 1997). In overproduction states, such as multiple myeloma, light chains, also known as Bence-Jones proteins, may produce nephrotoxicity. It was shown previously that free xcexa- and xcex-light chain isotypes bind to a single class of renal proximal tubular receptors which facilitate internalization and degradation (Batuman et al., 1997). To date, however, the receptor(s) which mediate endocytosis of light chains in the proximal tubule have not been characterized.
It has long been postulated that glycoproteins expressed at the apical pole of proximal tubule cells of the kidney acted as scavenger pathway receptors. The only known and cloned receptor until now is megalin, a fairly abundant proximal tubule protein, also known as gp330 or the xe2x80x9cHeymann antigenxe2x80x9d. Megalin is a classic single transmembrane domain giant glycoprotein receptor (Saito et al., 1994), which belongs to the LDLR family (Yamamoto, 1984), and is closely related to the xcex12-macrogilobulin receptor, which is not expressed in the kidney (Moestrup, 1994). Characterization of megalin revealed that, like the xcex12M receptor, it was a multiligand receptor. Of particular interest for the renal pathology, megalin binds tPA and urokinase in complex with the corresponding inhibitor, but is also a polybasic drug receptor, binding ligands such as the aminoglycoside antibiotics (Moestrup et al., 1995).
Thus the prior art is deficient in the lack of renal receptors for toxic, physiological, and pathological proteins and drugs (such as myeloma light chains) and more generally, components that may gain access to the proximal tubule fluid. Further, the prior art is deficient in the lack of effective means of preventing renal toxicity by utilizing renal binding proteins or fragments thereof for such components. The present invention fulfills these long-standing needs and desires in the art.
The present invention discloses renal binding proteins for ligands. Also disclosed are the potential uses of these proteins for therapy to prevent renal toxicity or other types of toxicity.
In one embodiment of the present invention, there is provided a DNA encoding a cubilin protein selected from the group consisting of: (a) isolated DNA which encodes a cubilin protein; (b) isolated DNA which hybridizes to isolated DNA of (a) and which encodes a cubilin protein; and (c) isolated DNA differing from the isolated DNAs of (a) and (b) in codon sequence due to the degeneracy of the genetic code, and which encodes a cubilin protein. Preferably, the DNA has the sequence shown in SEQ ID No. 1, and cubilin protein has the amino acid sequence shown in SEQ ID No. 2. Still preferably, the DNA is expressed in the tissues like kidney, spleen, brain, liver, heart and thyroid.
In one embodiment of the present invention, there is a vector capable of expressing the DNA adapted for expression in a recombinant cell and regulatory elements necessary for expression of the DNA in the cell. Specifically, the DNA encodes a cubilin protein.
In another embodiment of the present invention, there is a host cell transfected with the vector expressing a cubilin protein. Specifically, the host cell is selected from the group consisting of bacterial cells, mammalian cells, plant cells and insect cells. More specifically, the bacterial cell is E. coli. 
In another embodiment of the present invention, there is provided isolated and purified cubilin protein or fragment coded for by DNA selected from the group consisting of: (a) isolated DNA which encodes a cubilin protein or fragment; (b) isolated DNA which hybridizes to isolated DNA of (a) and which encodes a cubilin protein or fragment; and (c) isolated DNA differing from the isolated DNAs of (a) and (b) in codon sequence due to the degeneracy of the genetic code, and which encodes a cubilin protein or fragment. Preferably, the protein has the amino acid sequence shown in SEQ ID No. 2, and the fragment has amino acid sequence consisting of one or more of the sequences selected from the group consisting of SEQ ID Nos. 21-27.
In another embodiment of the present invention, there is provided a method of detecting expression of the cubilin protein or fragment in a sample, comprising the steps of: (a) contacting mRNA obtained from the sample with a labeled hybridization probe; and (b) detecting hybridization of the probe with the mRNA.
In another preferred embodiment of the present invention, there is provided a pharmaceutical composition comprising the cubilin protein or fragment and a pharmaceutically acceptable carrier. Such composition can be used for treating or reducing nephrotoxicity or other types of toxicity in an in-need individual.
In still another embodiment of the present invention, there is provided a receptor for a variety of ligands, comprising a cluster of EGF repeats and a cluster of CUB domains. Specifically, the receptor is cubilin and the ligand is selected from the group consisting of immunoglobulin light chain, myoglobin, intrinsic factor-vitamin B12, metallothionein, xcex2-2-microglobulin, amyloid, hemoglobin, haptoglobin, interferon, insulin, cytochrome c, lysozyme, transferrin, transthyretin, polybasic drugs, apolipoprotein AI, high density lipoprotein and receptor related protein. More specifically, a representative example of polybasic drug is gentamicin. Representative examples of immunoglobulin light chain include xcexa-light chain and xcex-light chain.
In still yet another embodiment of the present invention, there is provided a method of detecting renal damage by measuring the level of cubilin in the urine of an individual suspected to have such damage. If the urinary cubilin level is lower than that of a normal individual, the test individual might have chronic renal damage. On the other hand, the test individual might have renal damage of acute origin if the urinary cubilin level is higher than that of a normal individual.
Other and further aspects, features, and advantages of the present invention will be apparent from the following description of the presently preferred embodiments of the invention given for the purpose of disclosure.